Immunoassays utilize the specific binding capabilities of antibodies to detect the presence of target molecules in solution. Although the general principle is applicable to a broad range of problems, major commercial interest has centered on medical diagnostic applications for a wide variety of analytes in biological fluids such as blood, saliva, and urine.
Several types of immunoassays, useful for distinct applications, already exist. Each such assay type requires a way of distinguishing whether binding sites on an antibody are occupied or free. Typically this is accomplished by means of label such as an atom, molecule, enzyme or particle attached permanently to either the antibody or to an analog of the analyte.
Sensitivity and specificity are key parameters of an immunoassay. Specificity relates primarily to the antigen binding site of the antibody, which is inherent to selection of variable region gene segments and is independent of the assay configuration. Sensitivity relates primarily to the affinity of the antibody for its ligand(s) and to the inherent detectability of the label. For example, radioisotopes, used for radioimmunoassay, can be detected at significantly lower concentrations than fluorescent molecules. Enzyme labels are detectable at concentrations similar to fluorescent labels. When substrates that produce fluorescent or chemiluminescent products are used with enzyme labels, the sensitivity of resulting immunoassays is comparable or greater than with radioisotope labels.
Many conventional assay techniques are considered competitive in that the analyte and labeled component have comparable affinities of the antibody binding site. One example of such a competitive method is found in U.S. Pat. No. 3,817,837 by Rubenstein and Ullman which describes a technique in which ligand and enzyme-bound-ligand compete for antibody binding sites. Since binding of the antibody to the enzyme-bound-ligand alters its enzymatic activity, the concentration of ligand present can be estimated by measuring the rate at which such a mixture converts substrate to product.
Immunoassays can be further characterized as homogeneous and heterogeneous. In a heterogeneous method, the label is equally detectable in bound and unbound states. To obtain any meaningful assay results physical separation of the bound versus unbound antibody is required. A common strategy for accomplishing this separation entails associating the label to a solid phase which can be physically separated from the liquid phase prior to the detection step. A typical heterogeneous assay is the Tandem EIA from Hybritech, Inc.
In a homogeneous method, the detectable property of the label is inherently different depending on whether bound or unbound. In its bound state, the label will haver greater or lesser signal intensity. Usually, binding of antibody to the labeled ligand causes a decrease in signal intensity, e.g., when the label is an enzyme. Typical products in this category include the EMIT line of enzyme immunoassays from Syva Company and the TDX line of fluorescence polarization immunoassays from Abbott Diagnostics.
Two further characteristics of immunoassays are particularly noteworthy. These are the minimal concentration of analyte that can be detected, and the dynamic range of detection. The dynamic range is the range of analyte concentrations over which signal from a label changes from zero to maximum. The order in which the sample, the antibody, and a labeled component are combined can significantly affect both of these key parameters by affecting the degree of binding of the labeled component, which in turn affects detection of the label.
In certain known assay methods, the antibody and the analyte are combined prior to addition of the labeled component. In others, the analyte and labeled component are combined prior to addition of the antibody. Each of these cases requires providing two separate reagents that are combined with the sample containing the analyte. The need for two such separate reagents can be inconvenient and result in a more cumbersome, complex method. Moreover, because precise volumetric measurement of each reagent is critical to good assay performance, the necessity of two measuring steps can cause errors which may lead to distorted results.
One method to improve assay precision and thereby enhance assay sensitivity is to provide a premixed complex of the antibody and labeled component. This is problematic, however, because the binding reaction is generally found to be irreversible. Thus, when a complex of the labeled analyte and antibody are combined with a solution containing the analyte, no appreciable displacement of bound label occurs in a meaningful time frame (seconds to minutes).
The present invention relates to assay methodology that employs a complex of receptor and a ligand, wherein the receptor-ligand complex dissociates in the presence of an analyte and a stable receptor-analyte complex is formed. Dissociation of the receptor-ligand complex in the presence of analyte is detectable thereby positively indicating the presence of analyte in a test sample. Methods for designing, preparing, using, and stabilizing such complexes are taught. The methodology is applicable both to homogeneous assays and heterogeneous assays for analytes encompassing a broad range of types and sizes.